Dual Antibody Conjugated Drug Loaded Nanoparticle for Non-Small Cell Lung Cancer Treatment Using Targeted Immunotherapy

Introduction: Non-small cell lung cancer (NSCLC) is the leading type of lung cancer, which leads to 25% cancer-related death worldwide. Blocking of immune checkpoints by monoclonal antibodies, known as immunotherapy, has revolutionized the treatment landscape. One approach against NSCLC is to inhibit the interaction between program cell death protein 1 (PD1) present on the surface of T cells and program death ligand 1 (PDL1) present on tumor cell surfaces, thus activating the adaptive immune response. However, the majority of the patients do not respond to PD1-PDL1 immunotherapy. So, it is necessary to include additional treatment methods along with the traditional immunotherapy. Cancer cells express CD47, which interacts with signal regulatory protein alpha (SIRPα) in macrophages as an innate immune regulator. It blocks phagocytosis and polarizes the macrophages to protumorigenic M2 phenotype. Thus, the simultaneous blocking of CD47 and PDL1 may lead to the activation of both innate and adaptive immunity. Similarly, using pharmacological inhibitors to block specific oncogenic pathways is another popular approach, which has shown remarkable treatment outcomes with minimum side effects. Here, we demonstrate a combination therapy that can block both PDL1 and CD47 and simultaneously inhibit PI3K-AKT-mTOR oncogenic pathway.
Liposomal nanoparticles emerge as the potential platform due to the advantages of (i) easy conjugation of dual antibodies, (ii) higher drug-antibody ratio, and (iii) enhanced permeability and retention effect in tumors. Here, we developed a novel dual antibody conjugated drug-loaded nanotherapeutics (ADN) by combining anti-PDL1, anti-CD47 monoclonal antibodies and a dual PI3K/mTOR inhibitor drug PI-103 onto a lipid nanoparticle.

Materials and

Methods: ADNs were prepared by thin film hydration method using L-α-phosphatidylcholine (SOY-PC, 60 mole %), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG-COOH, 30 mole%) and drug PI103 (10 mole%). PI103 was conjugated with cholesterol hemisuccinate by coupling reaction with Ethyl-3-(3’-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxy succinimide (NHS) in presence of N,N-diisopropylethylamine base to yield PI103-Ch, which has shown increased stability in a lipid membrane. The characterization was carried out in 400 MHz 1H-Nuclear Magnetic Resonance Spectroscopy. The nanoparticles were conjugated with both anti-PDL1 (Anti-mouse PDL1 (B7-H1), Rat IgG2b, κ) and anti-CD47 (Anti-mouse/human/rat CD47, Mouse IgG1, κ) by EDC-NHS coupling reaction. We have used size exclusion chromatography using Sephadex G50, mechanical extrusion using 200 nm membrane and dialysis using 300 kDa membrane to purify the uniformly sized anti-CD47-PDL1-ADNs. The size of the particles was measured by dynamic light scattering (DLS), and the surface charge was studied by measuring zeta potential. The concentration of the total antibodies present in the ADN was calculated using a bicinchoninic acid assay, and the total drug concentration was calculated from the UV-Vis absorbance calibration plot. Enzyme-Linked Immunosorbent Assay (ELISA) was carried out using ADN, HRP-conjugated secondary antibodies and TMB substrate for readout. Biolayer Interferometry (BLI) analysis was carried out using a streptavidin sensor and biotinylated antigen with varying concentrations of nanoparticle solution. In-vitro cytotoxicity studies were performed using an MTT assay with mice and human lung cancer (LLC, A549) cells. The in-vivo efficacy studies were done in mice by injecting them with Lewis lung carcinoma (LLC) cells.

Results, Conclusions, and Discussions: Results and

Discussion: A novel dual antibody conjugated drug-loaded nanoparticle was designed and synthesized using lipids, antibodies anti-PDL1, anti-CD47, and cholesterol-conjugated PI103 drug (Fig.1a). The nanoparticles were characterized by DLS, and they were of size 145±5nm. The ADNs exhibit stability for up to 45 days (Fig.1b). The ADNs showed a drug-to-antibody ratio (DAR) of 308.9±9.8. This is indeed one of the greatest advantages of ADN over conventional antibody drug conjugates (ADC), which have DAR between 1-8. There is a fair chance that the antibodies will lose antigen-binding capacity upon conjugation. We proved that the antigen-binding capability of antibodies on ADN is still intact by using ELISA and observed a concentration-dependent readout confirming successful binding (Fig.1c). To understand the competency of ADN over respective monoclonal antibodies, we performed competitive ELISA. Interestingly, the majority of the ADNs were retained on the antigen-coated plate even after the addition of pure monoclonal antibody. In order to estimate the antigen binding quantitatively, the binding strength of ADN with respective antigens was studied by Bio-layer interferometry (BLI). A dissociation constant (Kd) of 32.7±0.12nM was obtained for ADN against the combination of CD47 and PDL1 antigen-coated plate, suggesting a strong affinity (Fig.1e). On a comparative study between the binding of ADN with PDL1+CD47 and PDL1 and CD47 separately using BLI, higher binding was observed for both antigens suggesting the synergistic binding of ADN with antigens (Fig.1e). The ADN also showed higher internalization in cancer cells when compared to no-antibody and single antibody-conjugated nanoparticles (Fig.2a), and this resulted in enhanced cytotoxicity (Fig.2b). The anti-CD47-PDL1-ADN treated mice group show increased antitumor efficacy over the untreated control group (Fig.2c).

Conclusions: In summary, a triple punch nanotherapeutic scaffold was unveiled, which can block both PDL1 and CD47 immune checkpoints and activate adaptive and innate immune responses simultaneously. Moreover, the ADNs can selectively deliver the drug to the cancer cells. Taken together, this ADN strategy scopes for a transformational change in the treatment of NSCLC, reducing the off-target toxicity and side effects.

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